Project Information

Summary:

PROJECT BACKGROUND
The Poruban Nursery is a small family-owned and operated nursery, founded in 1964 by Floyd and Ann Poruban, specializing in ericaceous plants, broad leaved evergreens, and companion plants. It occupies 24 acres of acid, well-drained sandy soils deposited during the last ice age, and has been at this location since 1969. Sustainable operating practices have been practiced for at least 25 years and include mostly hand weeding with subsequent low applications of herbicides, only spot applications of pesticides when needed, and use of nutrition to control plant diseases. These practices have resulted in a huge array of predatory insects and beneficial soil microorganisms, a vibrant population of vertebrates, and healthy nursery stock. Sustainable practices have permitted the Clavaria fungi to prosper and become prominent in our fields. This project has allowed harnessing of this rather rare resource and will, hopefully, allow it to be controllable and usable by the horticulture industry.

This project was designed to happen in three logical phases. Phase I was designed to verify ericoid mycorrhizal associations in vitro to establish a sound basis for plant trials. Phase II involved developing a reproducible growth medium and testing several materials that could be used as commercially usable spawn. Since Clavaria fungi do not produce spores in culture, alternative spawn production methods to those currently used for other mycorrhizal inocula had to be developed. Commercially usable in this case is defined as easily produced, uniform, quantifiable, easily used, and containing viable mycelium. Techniques for producing Clavaria inoculum were adapted from commercial mushroom spawn procedures, and the underlying nutrient medium was loosely based on Modified Melin-Norcrans (MMN) medium, currently used for mycorrhizal experimentation.

Phase III will be implementation and testing of Phase II mycorrhizal inoculum under commercial conditions at the Poruban Nursery and then at other nurseries and small fruit growers. This phase will require a separate grant, but is included here for context. Phases I and II, only were covered by this grant,

PROCESS
Phase I: Once one Clavaria species was purified, in vitro mycorrhizal establishment could be performed. Vaccinium macrocarpon was the model ericoid host, and Clavaria acuta was the mycorrhizal inoculum. V. macrocarpon was used because the seeds are easily obtained and easily sterilized due to a smooth seed coat. Clavaria acuta, a species originally isolated from basidiocarps under Taxus x medis and Platanus x acerifolia, unusually endomycorrhizal species in their respective groups, was used because it was the first species purified. C. acuta was also seen under Malus spp. at the Poruban Nursery in 1996. The assumption was that for mycorrhizal formation, fungal species does not matter and that only fruiting characteristics are influenced by the host-fungus interaction.

The goal of Phase I was to establish a mycorrhizal relationship in vitro. The agar medium used for mycorrhizal studies is usually MMN medium (Figure 1), but a new, less soluble nutrient medium was used in this experiment (Figure 2). [Editor’s note: For copies of the figures mentioned in this report, please contact NCR-SARE at: ncrsare@umn.edu]

The nutrients in MMN medium are all water soluble and all immediately available to both fungi and plants. Unfortunately, however, any foliar response that would indicate mycorrhizal association would be undetectable because the plant could absorb all nutrients independently from the fungus. A medium with mostly insoluble nutrients is more conducive to an indicative foliar response that would time root harvest.

For nutrients to be absorbed by the plant, solubilization by the fungus would be necessary, and a more accurate foliar response would be elicited. Several ingredients in MMN were soluble chlorides. It was determined that there was more chlorine than nitrogen, calcium, magnesium, sulfur, iron, or sodium. Chlorine is detrimental to most microorganisms and most plants, and it did not make sense to use a medium where chlorine was the third highest concentration after phosphorous and potassium. Other nutrient concentrations were changed to more closely reflect relative nutrient concentrations in a general fertilizer solution. Although further testing and modification is necessary to test fungal toxicity limits and make the medium more insoluble, especially regarding nitrogen, the new medium worked well for this experiment. Duddridge (1986) found that no carbohydrate source should be added to the agar medium so any required carbohydrate source must come from the seedling. This promotes fungus-root interaction. The in vitro procedure used was developed by J Duddridge (1986). Only seedling roots are actually maintained in the strictly controlled in vitro environment, while the top is exposed to air in a humidity chamber. Good technical proficiency in sterile technique and use of a laminar flow hood are essential to the success of this procedure.

The procedure was as follows: Sterile seedlings were sprouted on 1.5% w/v water agar in sealed test tubes (Figure 3) under fluorescent lights. Poruban’s medium (Figure 2) was sterilized in a pressure canner for 15 minutes at 15 lbs. pressure, was allowed to cool slightly and was poured approximately 5 mm deep into 100 mm x 10 mm plastic petri dishes and allowed to solidify. For each plate, a cotton swab was used to apply 70% isopropanol to the lid side and petri dish side and allowed to evaporate. At the alcohol application site, a V-notch was cut into the lid with a red-hot scalpel heated with a Bunsen burner, and a corresponding notch was cut into the plate to the agar surface (Figure 4). When aligned, the notches formed a diamond pattern (Figure 5). A small dollop of USP grade lanolin was applied to the plate notch and a 3-4 cm seedling was pressed into the lanolin until the roots touched the agar. Because the seedlings were curly, the roots were pressed into the agar to maintain contact (Figure 6). Fungal inoculum was placed on the agar surface 5 to 10 mm away from the root system. Another lanolin dollop was applied to the cover notch, the notches were aligned, and the plate was sealed with ½” electrical tape (Figure 7).

Finished plates are placed into a growth chamber under high humidity with fluorescent illumination at room temperature. In this experiment, six inoculated plates were stood on edge in a plastic cookie box containing a small dish of warm water (Figure 8) then placed under lights, No uninoculated control plates were used in this experiment because indicative foliar response was not the primary concern, although it was useful in determining when to sample roots for mycorrhizae. Plants were incubated for 2 weeks before photographing.

After the plates were photographed, 4 plates were dismantled, and 2 were left to continue in the incubator. The roots were severed from the plated seedlings and from uninoculated water-agar seedlings. The entire root system of each was placed in a different test tube containing formalin-acetic acid-alcohol solution then stained with chlorazol black E using the procedure form Brundrett et al. (1996), fixed on microscope slides, and then photographed.

Phase II
Various bulk media were tested to determine their suitability to support Clavaria growth. Seventeen media were selected based on either current use in plant propagation and small pots or potential use as after-market inoculum. While this does not span the entire range of potential media, it represents a broad range of natural and synthetic materials. It was unnecessary to include vermiculite because of its extensive use in early mycorrhizal studies as an in vitro medium. Figure 9 shows the media, which are labeled as follows: Row 1 (L-R) Pin Cherry twig, Isolite®, Grodan® cubed rock wool, cotton clothesline, and Black Willow twig; Row 2 (L-R) 3M Scotchbrite® scratcher pad, loose rock wool; Row 3 (L-R) Fafard® peat moss, Oasis® floral foam, PVP® perlitle, ericaceous field soil, and coir fiber; Row 4 (L-R) Waterworks® water gel, beer barley, masonry sand, bleached notebook paper; Bottom Swheat Scoop® wheat cat litter.

Seven samples of each medium were put into 16 mm culture tubes and soaked with liquid medium as described in Phase I, but without agar. Tubes were capped with vented plastic caps and cooked in a pressure canner for 15 minutes at 15 lbs pressure. Each tube was inoculated with Clavaria acuta and incubated at approximately 30ºC for 2 weeks before photographing.

PEOPLE
Many people helped with this project, and deserve to be acknowledged. This first are Floyd and Ann Poruban, my parents, and Debora Poruban, my loving wife. Dad taught me all the skills needed to construct and repair lab apparatus, helped build a suitable work space, and provided financial support as well as manuscript editing. Mom provided kitchen table space, to expedite research before a more suitable work space could be built, financial support, and manuscript editing. My wife allowed me precious free time by covering many of my home chores, provided organizational and technical support and computer graphic assistance, procured supplies, and helped edit the manuscript. Without my family, this project could not have been completed.

Dr. Pierluigi Bonello, Department of Plant Pathology, The Ohio State University, shared his expertise, research tools and procedures, and library access. He has also provided research critiques and kept me focused.

Dr. Mark Starrett, University of Vermont, provided the staining procedure and expertise on how to use it effectively.

The Groden Corporation and the Oasis Corporation provided free samples of their products to test.

Dr. Martina Veigl, and Dr. David Sedwick, Case Western Reserve University, donated several key pieces of equipment including a balance, incubator and sterilizing oven, without the research would have been difficult, if not impossible.

Dr. Jim Chatfield and Dr. Charles Behnke from The Ohio State University Cooperative Extension Service wrote letters of support.

The North American Heather Society and Northeast Heather Society both provided financial support to the initial mycorrhizal tests on heathers, which provided the basis to proceed with this experiment. They provided funding for the first opportunity to synthesize Clavaria mycorrhizae in the greenhouse and permitted research observations to continue when family finances were impossibly tight.

For the opportunities and faith offered by these and many other unnamed individuals who extended good wishes, thank you!

RESULTS
Phase I
Clavaria grew slowly from the inoculation point to the seedling root system. It took 2 days to grow 5-10 mm, but when the mycelium made contact with seedling roots, the dishes were completely ramified with mycelium within 2 more days, a surprising development! Clavaria was vigorous, covered the roots with a profuse mat, and eventually formed rhizomorphs. Rhizomorphs are root-like structures formed by twisted strands of fungal hyphae, are a sign of good vigor, and they support fruiting structures in basidiomycetes (Stamets, 2000).

Seedling roots were difficult to clear and most uninfected roots retained a transparent brownish orange phenolic color (Figure 10). A perfectly cleared V. macrocarpon root is shown in Figure 11. Mycorrhizae formed profusely in V. macrocarpon roots. Fungal hyphae were stained black. It is will known that ericoid mycorrhizae form in root cortex cells and form a false mantle at the root surface. Though individual mycorrhizal structures are not clearly visible due to incomplete root clearing, mycorrhizae are evident by dark staining in the root tissue and heavy root surface fungal growth. Epidermal cells appear clear, but mycorrhizal root cortex regions are opaquely black (Figures 12-16).

Seedling foliage color changed from yellow to reddish, a typical color for healthy V. macrocarpon seedlings (Figure 17). The two intact mycorrhizal plates continue to grow with no apparent decline in the seedling health. For a quick anecdotal test, a seventh plate was made 2 weeks after the initial 6 and was purposely allowed to be contaminated by aerial spores. The contaminated plate was placed in the same incubator as the incubated ones for 2 weeks. A comparison between plants in an inoculated plate and the contaminated plate appears in Figure 18. The incubated seedling at approximately 1 month incubation time was still vigorous and red, and the contaminated seedling was withering, dropping leaves and browning. It is apparent from all collected data that Clavaris mycorrhizae formed in inoculated plates, are not detrimental to the V. macrocarpon seedlings, and are beneficial. However, true benefit will only be determined from extensive long term controlled studies in Phase III. Clavaria acuta was collected from basidiocarps under Taxus spp. and Platanus x acerifolia. Therefore, the assumption that Clavaris species is irrelevant to ericoid mycorrhizae formation has been supported.

Phase II
Most media supported Clavaria growth, however no growth was observed on the scratcher pad, on masonry sand, or on peat moss. The results are in Table 1. Photos of each group of medium tubes are in Figures 19-34. Some groups have less than 7 tubes due to breakage, and, unfortunately, the slide for the willow twigs was destroyed. Rhizomorphs formed on paper (Figure 35) and perlite. Slow growth was initially observed on the bleached paper, cubed rock wool, cotton clothesline, and willow twig. It was estimated that the growth on these media was about 2 weeks behind the other media, but ultimate growth overtook the others in time.

DISCUSSION AND CONCLUSIONS
Unfortunately, Phase I and II took 2 years to complete due to unforeseen contamination of existing primary cultures. Clavaria basidiocarps only sprout once per year in October, and it took the first year to develop a better isolation medium to separate Clavaria from contaminants while allowing a second chance to isolate from the wild, if necessary. Clavaria argillacea has long been a known companion of ericaceous plants, and Clavaris acuta has been seen frequently in containers of evergreens in conservatories (Watling et al, 1991), but has not been mentioned in the literature associated with ericaceous plants.

This project provided some minor technical challenges. The light banks were an easy task with some old computer desk legs and fluorescent fixtures (Figure 36), but building the Bunsen burner apparatus without it exploding was a greater challenge. A few parts from Sears, old barbeque controls, a propane tank, and some creativity worked perfectly (Figure 37). Once all the minor difficulties were solved, the research was accomplished without incident.

Phase I provided strong evidence for Clavaria mycorrhizae formation in vitro. More trials and better controls must be conducted to prove that foliar response is absolutely due to mycorrhizal infection, and more cellular detail would have been preferred, but otherwise it went flawlessly.

V. macrocarpon roots proved extremely difficult due to high phenolic content, and more attempts at root clearing and staining might provide clearer structural details within cells. The staining procedure must be modified to accommodate the phenolic content but not disintegrate roots by overcooking.

The fungal growth response after root contact was the most surprising observation in Phase 1. Fungal growth was accelerated by plant interaction and could provide evidence for less host specificity than indicated by fruiting response in basidiomycetes. If a hypothesis is made that fungal fruiting response is present on ideal mycorrhizal hosts and is absent on less ideal hosts, this experiment provides a sound basis for studying host dependent fruiting responses independent from mycorrhizal affiliation. Fungal-host interactions should produce improved fungal and plant growth responses on ideal hosts. A possible explanation for increased fungal vigor in this case could be ascorbic acid. Since V. macrocarpon produces fruit with high ascorbic acid content, it is not inconceivable that ascorbic acid – a common chelating agent for iron – is also exuded from roots and allows for greater iron uptake by the fungus. Iron helps certain fungi grow in anaerobic conditions (Garroway, et al. 1991), such as those in soil or in sealed Petri dishes.

Clavaris acuta basidiocarps, from which the experimental inoculum was cultured, were collected from Taxus spp. and Platanus x acerifolia, yet the fungus formed a mycorrhizal relationship with V. macrocarpon. No literature citations list C. acuta basidiocarps associated with ericaceous plants. From this, it is apparent that C. acuta is a versatile fungus with a wider host range than previously indicated by basidiocarp formation. This is the first research that has shown that C. acuta could be associated with anything but conifers. Trials with C. acuta should be conducted with Taxus spp., Platanus spp., and Malus spp. in vitro.

Claveria argillacea normally is mycorrhizal on, and fruits profusely with ericaceous plants (Mueller et al, 1985; Watling, 1991), and if it is like C. acuta, should have a wider host range. Inoculation and media trials using purified C. argillacea in vitro should show increased fungal and plant growth response over C. acuta on ericaceous hosts. Greenhouse inoculation trials at the Poruban Nursery in 2002 with Clavaria argillacea on heathers resulted in mycorrhizal formation, but the trials were in perlite in a less controlled environment and severe mortality caused by excessive heat and dryness prevented study of plant growth responses.

Phase II demonstrated that C. acuta could grow on a wide array of materials that could be used for commercial and consumer inoculation of host species. Media used can be classed into different groups based on origin. The cherry twig, willow twig, and paper contain undecomposed wood fiber with different secondary compounds. Peat moss and coir fiber (coconut hull fiber) are both partially decomposed plant fibers. Masonry sand and sandy ericaceous field soil both contain fine sand and are separated only by organic matter content. Rock wool and perlite are treated and reconstituted but chemically unaltered mined mineral products. Isolte, floral foam, scratcher pad, and water gel are totally synthetic materials. Beer barley and wheat cat liter are both high-starch grains. Cotton clothesline is a combination product containing plant fiber surrounding a synthetic fiber core and treated with a whitening coating.

Materials containing high starch and phenolic compounds generally supported more vigorous growth of C. acuta, however all but 3 materials could be used effectively as inoculum. An unexpected finding was that peat moss did not support C. acuta growth but coir supported vigorous growth. Peat moss is a frequent ingredient in ericaceous potting and propagating media. This study correlates with Linderman (2003). Linderman found that certain peat moss types selectively inhibit mycorrhizal fungi but that coir fiber was not inhibitory (Linderman et al, 2003).

The grain based media supported vigorous growth. Grain based media are used for mushroom spawn production (Stamets, 1983).

Waterworks water gel supported vigorous C. acuta growth. This finding correlates with Kuek et al. (1992) who used hydrogel to culture ectomycorrhizae for inoculating eucalypts. Growth did not continue to the tube bottom probably due to hypoxic conditions.

Cherry supported more vigorous early growth than willow, possibly due to differences in phenolic or salicylic acid, but differences decreased with time. Clavaria spp. has been observed growing on cherry wood in the wild in 1995 at the Poruban Nursery, but isolation was not pursued.

Floral foam supported vigorous fungal growth successfully on 1/7 of the trails. Further tube inoculum observations showed that the other 6 collections from hyphal margins on the original petri dish were too marginal and did not contain viable mycelium.

Ericaceous field soil supported vigorous mycelial growth when soaked with nutrient medium. Ericaceous roots within the soil apparently provide beneficial compounds even when sterilized, possibly a good indicator of mycorrhizal fungus attraction. Masonry sand, even when soaked with nutrient medium like other inert media, did not support fungal growth. Factors, including lack of composted organic material, tight structure, and lack of microbial secondary metabolites could be responsible for lack of growth.

Of the media that supported fungal growth, perlite and paper were the only media where rhizomorphs formed. While no discernable relationship between the compositions and suitability of paper and perlite is readily apparent, they obviously provide the best environments for growth of fruiting structures due to rhizomorph formation (Stamets, 2000). This is an interesting finding and should be explored further.

The scratcher pad did not support fungal growth, but no explanation of this exists at this time. It is not known whether any antimicrobial compounds are used in its manufacture.

BIBLIOGRAPHY
Bannister, P. and W. M. Norton. 1974. The response of mycorrhizal and non-mycorrhizal rooted cutting of heather to variations in nutrient and water regimes. New Phytologist 73, 81-89.

Duddridge, J. A. 1986. The development and ultrastructure of ectomycorrhizas IV. Compatible and incompatible interactions between Suillus grevillei (Klotzsch) Sing. and a number of ectomycorrhizal hosts in vitro in the presence of exogenous carbohydrate. New Phytologist 103(3), 465-471.

Powell, Conway L. 1982. The effect of the ericoid mycorrhizal fungus Pezizella ericae on the growth and nutrition of seedlings of blueberry (V. Corymbosum). Journal of the American Society for Horticultural Science 107(6), 1012-1015.

Pyatt, F. B. 1975. Clavaria argillacea on a spoil tip in south west England. Transactions of the British Mycological Society 64(1), 171.

PROJECT IMPACTS
All the media and nutrients used in this research are inexpensive enough to be used as plant inoculum in the propagation and small pot stages of ericaceous plant production, and C. acuta has shown remarkable adaptability to grow vigorously on most media as well as utilize insoluble nutrients. C. acuta and C. argillacea are both similar basidiomycete ericoid mycorrhizal fungi, and preliminary tests before this project have shown that they grown much more vigorously than competing ascomycete species on inorganic agar media. Ascomycete ericoid mycorrhizal fungi have proven to be slow in growing and commercially unfeasible on known media. Although it has yet to be proven if Claveria spp. is commercially useful, this project has provided enough evidence to warrant expanded trials and test it on a larger scale.

Positive commercial implication include more uniform and more vigorous young ericaceous plants, increased yields of useful characteristics later in production life, fewer disease problems, less mortality in propagation, and almost no waste of soluble, leachable chemical fertilizers in production. Uniformity, yield, mortality, and wasted resources are the most costly factors to control by current production means, and represent the most profitable source of savings if even partially controlled by mycorrhizae. No economic data has been found regarding the impact of wild ericoid mycorrhizae in field plant and fruit production operations, and no one has determined the impact of introduced mycorrhizal species on any form of ericaceous plant production in North America because none have been commercially available. Anecdotal forestry and horticultural sources either praise mycorrhizae as a useful tool, or claim no useful impact, but non-standard test methods and lack of controls preclude any useful determinations. Only larger scale long term production trials at several sites will provide economic impact data for Clavaria mycorrhizae. This project provides sound basis for expanded controlled trials in Phase III.

OUTREACH
I have been asked to speak at the Ohio Chapter of the International Society of Arboriculture Meeting in February 2006 where a slide show from this paper, including additional pictures, will be presented. A talk for the heather societies will also be arranged at a meeting sometime in September 2006. The concerns from this research will be addressed in the next 2 months and a paper will be published. Results from this research will be presented on the Poruban Nursery website at www.porubannursery.com, but due to technical difficulties, will not be presented until 2006. Advertising efforts will be made after Phase III.

Current outreach efforts have simply been personal phone calls with ericaceous plant propagators and liner producers, who have been unbelievably supportive and eager to test ericoid mycorrhizal fungi in their production settings. The market has been clamoring for ericoid mycorrhizal inoculum for years, and I have no doubt that they will tell their friends. Personal phone calls have been an extremely effective way to disseminate information about this project, and will continue. At least 3 ornamental nursery propagators have expressed interest in helping with this project, and small fruit propagators will be contacted shortly. Once refined, this project will be presented for publication in several specialty society journals and trade publications, including the American Rhododendron Society, American Azalea Society, Northeast Heather Society, North American Heather Society, and the Ohio Nursery and Landscape Association. Specialty small fruit publications will be contacted as they are located.

Cooperators

Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the author(s) and do not necessarily reflect the view of the U.S. Department of Agriculture or SARE.

Primary Sidebar

Footer

1122 Patapsco Building | University of Maryland | College Park, MD 20742-6715

This site is maintained by SARE Outreach for the SARE program and features research projects supported by the National Institute of Food and Agriculture, U.S. Department of Agriculture. SARE Outreach operates under cooperative agreement award No. 2017-38640-26822 with the University of Maryland to develop and disseminate information about sustainable agriculture. USDA is an equal opportunity provider and employer.